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| Status |
Public on Nov 16, 2017 |
| Title |
NRAS metastatic melanoma ChIP-BRD4 JQ1 (+) [SKmel147] |
| Sample type |
SRA |
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| Source name |
Cell culture
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| Organism |
Homo sapiens |
| Characteristics |
cell line: SKmel147
passage: N/A
antibodies: BRD4 (Bethyl Laboratories, A301-985A)
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| Treatment protocol |
treated with 10nM JQ1 (JQ1(+)) for 6 hours
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| Growth protocol |
grown in DMEM supplemented with 10% FBS, penicillin and streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For BRD2, BRD4 and MED1, 72x10^6 cells, were crosslinked with 1% PFA for 10 min at RT. ChIP was performed essentially as previously described (Roe, J.S., et al., BET Bromodomain Inhibition Suppresses the Function of Hematopoietic Transcription Factors in Acute Myeloid Leukemia. Mol Cell, 2015. 58(6): p. 1028-39).
Libraries for ChIP-seq were done as previously described (Hasson D., et al., The octamer is the major form of CENP-A nucleosomes at human centromeres. Nat Struct Mol Biol. 2013 Jun;20(6):687-95).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
ChIP-BRD4 JQ1 (+)
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| Data processing |
Native ChIP-seq: Reads were trimmed from Illumina adapters using in-house script. Reads were aligned using Bowtie (version 0.12.7) with parameters –l 40-65 –n 2 –S –best –k 1 –m 20. BAM files were generated using SAMtools. bigwig files were generated from BAM files using deepTools bamCoverage (version 2.4.1) with parameters –normalizeUsingRPKM –binsize 10
XL ChIP-seq: Reads were trimmed from Illumina adapters using in-house script. Reads were aligned using Bowtie (version 0.12.7) with parameters -k 1 -m 1 --best -S -n 2 -l 65 -q. BAM files were generated using SAMtools. duplicated reads were removed using SAMtools. bigwig files were generated from BAM files using deepTools bamCoverage (version 2.4.1) with parameters –normalizeUsingRPKM –binsize 10. Significant peaks were called using MACS2 callpeak (version 2.1.1.2) with parameters -f BAM -g 2.7e9 -s 100 --bw 200 —slocal 1000 -q 0.01.
ATAC-seq: Reads were trimmed from Illumina adapters using in-house script. Reads (40bp Paired-end) were aligned using Bowtie2 (version 2.1.0) with parameters -q -X 2000. BAM files were generated using SAMtools. Reads that align to mtDNA, with quality value Q<30, as well as duplicated reads, were discarded using in-house scripts and Samtools.. bigwig files were generated from BAM files using deepTools bamCoverage (version 2.4.1) with parameters –normalizeUsingRPKM –binsize 10. Significant peaks were called using MACS2 callpeak (version 2.1.1.2) with parameters –nomodel –nolambada –keepdup all –slocal 10000
RNA-seq: Reads (50bp, Paired-end) were aligned using TopHat (version 2.1.0). Transcriptome assemblies in FPKM, and differential expression ratios were computed with Cufflinks (version 2.1.1). In all samples, genes were called ‘expressed’ if normalized FPKM≥1.5. Genes were called downregulated upon JQ1 treatment if nFPKM≥1.5; log2FoldChange≤-0.625; and Padj<0.05. Genes were called overexpressed over NHM if nFPKM≥1.5 (in the melanoma sample); log2FoldChange≥2; and Padj<0.05. Genes were called downregulated upon AMIGO2 depletion if nFPKM≥1.5 (prior to depletion); log2FoldChange≤-1.2; and Padj<0.05. log2FoldChange≥1.2 for upregulated genes.
SEs calling: Enhancers (TEs) and super enhancers (SEs) were called based on ChIP-seq BRD4 enrichment in SKmel147 using the ROSE (Rank Ordering of Super-Enhancers) algorithm (stitching distance 12.5Kb and TSS exclusion zone size 2.5Kb. BRD4 levels were normalized to Input control; ChIP-Input)
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: fastq - raw illumina reads. bigwig - aligned reads pileup. diff - gene expression in normalized FPKM
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| Submission date |
Feb 03, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Emily Bernstein |
| E-mail(s) |
emily.bernstein@mssm.edu
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| Phone |
(212) 824-9335
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| Organization name |
Mount Sinai School of Medicine
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| Department |
Oncological Sciences
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| Lab |
Bernstein Lab
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| Street address |
1470 Madison Avenue, 6th floor Rm 302
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10029 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE94488 |
Harnessing BET inhibitor sensitivity reveals AMIGO2 as a melanoma survival gene. |
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| Relations |
| BioSample |
SAMN06294939 |
| SRA |
SRX2537518 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2476359_SKmel147-BRD4-JQ1_plus.bw |
412.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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